PDBsum entry 3mh2

Transferase

PDB id: 3mh2

Contents

Protein chain

330 a.a. *

Ligands

BOG

Waters ×40

* Residue conservation analysis

PDB id:

3mh2

Name:

Transferase

Title:

Mutagenesis of p38 map kinase establishes key roles of phe169 in function and structural dynamics and reveals a novel dfg-out state

Structure:

Mitogen-activated protein kinase 14. Chain: a. Synonym: map kinase 14, mapk 14, mitogen-activated protein kinase p38 alpha, map kinase p38 alpha, cytokine suppressive anti-inflammatory drug-binding protein, csaid-binding protein, csbp, max-interacting protein 2, map kinase mxi2, sapk2a. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: csbp, csbp1, csbp2, cspb1, mapk14, mxi2. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.30Å R-factor: 0.247 R-free: 0.318

Authors:

H.V.Namboodiri,M.Karpusas,M.Bukhtiyarova,E.B.Springman

Key ref:

M.Bukhtiyarova et al. (2007). Mutagenesis of p38alpha MAP kinase establishes key roles of Phe169 in function and structural dynamics and reveals a novel DFG-OUT state. Biochemistry, 46, 5687-5696. PubMed id: 17441692

Date:

07-Apr-10 Release date: 21-Apr-10

Supersedes:

2pto

Protein chain

Q16539  (MK14_HUMAN) -  Mitogen-activated protein kinase 14 from Homo sapiens

Seq: 360 a.a.

Struc: 330 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.7.11.24 - mitogen-activated protein kinase.
• Reaction:
  1. L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] + ADP + H⁺
  2. L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] + ADP + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

Biochemistry 46:5687-5696 (2007)

PubMed id: 17441692

Mutagenesis of p38alpha MAP kinase establishes key roles of Phe169 in function and structural dynamics and reveals a novel DFG-OUT state.

M.Bukhtiyarova, M.Karpusas, K.Northrop, H.V.Namboodiri, E.B.Springman.

ABSTRACT

In order to study the role of Phe169 in p38alpha MAP kinase structure and function, wild-type p38alpha and five p38alpha DFG motif mutants were examined in vitro for phosphorylation by MKK6, kinase activity toward ATF2 substrate, thermal stability, and X-ray crystal structure. All six p38alpha variants were efficiently phosphorylated by MKK6. However, only one activated p38alpha mutant (F169Y) possessed measurable kinase activity (1% compared to wild-type). The loss of kinase activity among the DFG mutants may result from an inability to correctly position Asp168 in the activated form of p38alpha. Two mutations significantly increased the thermal stability of p38alpha (F169A DeltaTm = 1.3 degrees C and D168G DeltaTm = 3.8 degrees C), and two mutations significantly decreased the stability of p38alpha (F169R DeltaTm = -3.2 degrees C and F169G DeltaTm = -4.7 degrees C). Interestingly, X-ray crystal structures of two thermally destabilized p38alpha-F169R and p38alpha-F169G mutants revealed a DFG-OUT conformation in the absence of an inhibitor molecule. This DFG-OUT conformation, termed alpha-DFG-OUT, is different from the ones previously identified in p38alpha crystal structures with bound inhibitors and postulated from high-temperature molecular dynamics simulations. Taken together, these results indicate that Phe169 is optimized for p38alpha functional activity and structural dynamics, rather than for structural stability. The alpha-DFG-OUT conformation observed for p38alpha-F169R and p38alpha-F169G may represent a naturally occurring intermediate state of p38alpha that provides access for binding of allosteric inhibitors. A model of the local forces driving the DFG IN-OUT transition in p38alpha is proposed.